synthesis, antimicrobial potential and toxicological activities ...mefloquine is a white or slightly...

ISSN: 0973-4945; CODEN ECJHAO

E-Journal of Chemistry

http://www.ejchem.net 2012, 9(4), 2245-2254

Co(II) Complex of Mefloquine Hydrochloride:

Synthesis, Antimicrobial Potential, Antimalaria and

Toxicological Activities

ADEDIJI J. FEMI1*

AND OBALEYE J. AYOOLA2

1Department of Chemistry, Federal University of Agriculture Abeokuta, P.M.B. 2240,

Abeokuta, Ogun State, Nigeria

2Department of Chemistry, University of Ilorin, P.M.B. 1515, Ilorin Kwara State

[email protected]

Received 4 April 2011; Accepted 2 June 2011

Abstract Transition metal complex of Co(II) with Mefloquine hydrochloride

(antimalaria drug) was synthesized using template method. Chemical analysis

including conductivity measurements and spectroscopic studies were used to

propose the geometry and mode of binding of the ligand to metal ion. From

analytical data, the stoichiometry of the complex has been found to be 1:1.

Infrared spectral data also suggest that the ligand (mefloquine hydrochloride)

behaves as a tridentate ligand with N:N:O donor sequence towards the metal

ion. The complex generally showed octahedral coordinate geometry.

Conductivity measurement of 10-2 mol dm-3 methanol solution of the complex

indicated non-electrolytic nature of metal complex. It also revealed that the

ligand anions were covalently bonded to the complex. In-vivo evaluation of

antimicrobial studies of the metal complex showed greater activities when

compared to the free mefloquine.The complex was screened against malarial

parasites (Plasmodium yoelii nigeriensis): It was evident from the results

obtained that Co(II) mefloquine has highest clearance of about 80%

parasitaemia reduction compared to the free mefloquine. The ligand and

metal complex were screened for their toxicological activities at the dose of

0.60 mg/Kg body weight twice daily for seven days on the alkaline

phosphatase (ALP), alanine aminotranferase (ALT), and aspartate

aminotransferase (AST) activities of rat serum, liver and kidney. Overall, it

was revealed that both mefloquine and its metal complex do not showed

toxicity particularly on the liver and kidney.

Key words: Transition metal, antimicrobial, antimalaria activity, toxicological studies.

Introduction

Complexation chemistry is quite simply the chemistry of coordination compounds

containing a central atom or ion to which are attached molecules or ions whose number

ADEDIJI J. FEMI 2246

usually exceeds the number corresponding to the oxidation number or valence of the central

atom or ion. They are of great theoretical importance and they are also of great practical

utility as well1.

Over the past three decades, intensive efforts have been made to design novel

compounds to confront new strains of resistance micro-organisms. The ongoing intense

search for novel and innovative drug delivery systems is predominantly a consequence of

the well established fact that the convectional dosage forms are not sufficiently effective in

conveying the drug compound to its site of action and this have necessitated the needs to

search for more potent drugs. The recognition of the potential employment of metal

complexes and chelates in therapeutic application provides useful outlet for basic research

in transition metal chemistry 2,3,4,5

.

Mefloquine Hydrochloride (Ligand employed in this study) is (±)-erythro-α-(2-

piperidyl)-2,8-bis(trifluoromethyl)-4-quinoline methanol, and it is known for antimalaria

activity. The choice of quinoline moiety was as a result of the success with the case of

chloroquine. Mefloquine was the only candidate drug that came off successfully during

Vietnam War. Its total synthesis was first reported by Ohnmacht et al.6. More than 10,000

synthesized compounds, most of which were based on the quinoline moiety, were screened

for antimalaria activity during the Vietnam War at the Walter Reed Army Institute (WRAI)

in U.S.A7. Mefloquine is a white or slightly yellow, crystalline powder, very soluble in

water, freely soluble in methanol and alcohol. It melts at about 260 °C with decomposition.

It shows polymorphism. Since the ligand (mefloquine) consists of potential binding sites

such as oxygen and two nitrogen atoms, this work set out to study the coordination

tendencies, characterization after complexing with metal and the biological activities.

Experimental

Material

Metal salt, Cobalt (II) chloride hexahydrate used for the Complexation was obtained from

British Drug Houses chemical limited, Poole, England and was used as supplied. The

ligand (mefloquine hydrochloride) was obtained from SWISS pharmaceuticals Company

Lagos, Nigeria. ALP, ALT, and AST assay kits were obtained from Randox Laboratories

Limited, Antrim, United Kingdom. Isolates of Escherichia coli, Klebsiella pneumonia and

staphylococcus aureus were obtained from the Department of Microbiology, University of

Ilorin, Nigeria. Albino rats (Wistar strain) were obtained from the Department of

Biochemistry, University of Ilorin, Ilorin, Nigeria. Plasmodium yoelii nigeriensis used in

this study were obtained Through University of Ibadan Teaching Hospital. Swiss mice,

obtained from the Department of Biological sciences, University of Ilorin, Nigeria. This

study was carried out in the Department of Chemical Sciences Laboratory, Ajayi Crowther

University, Oyo. Nigeria.

Infra-red spectra of the ligand and complex were recorded in KBr disc in the range

4000 - 600 cm-1

on PUC Scientific model 500 FTIR Spectrometer. Electronic spectra were

done on Aquamate Spectrophotometer Model V4.60. The metal estimation was done using

an Alpha4 Atomic Absorption Spectrophotometer with PM 8251 simple-pen recorder.

Co(II) Complex of Mefloquine Hydrochloride

2247

Conductivity measurements were carried out using WTW Conductometer Bridge. Thin

layer Chromatography was carried out using TLC plate coated with silica gel.

Synthesis of the Metal Complex

The complex was prepared based on previous reported procedures with slight modifications 1,4

. 0.01mol of ethanolic solutions of Cobalt(II) chloride (CoCl2.6H2O) were prepared in a

round bottomed flask. 0.01mol (4.148g) of mefloquine hydrochloride was dissolved in 20

cm3 ethanol and added to the solution of the metal salt in 10 cm

3 ethanol in a round-

bottomed flask fitted with a condenser and refluxed with constant stirring for 2 h. The

chelate were separated out after leaving it for four days. The metal chelates thus separated

were filtered and washed with methanol and then with distilled water to remove unreacted

ligand and metal. Finally the solid complex was dried in a dessicator. 10% methanolic

ammonia (buffer) solution was used to maintain the pH of the reacting solution of metal

salt and ligand under reflux.

Antimicrobial Screening of the Ligand and Metal Complex

The stimulatory or inhibitory activity of the ligand and the metal complex synthesized were

determined according to the procedure previously reported by Obaleye and Famurewa8 as

modified by Mohamed and Abdel-Wahab 9. The bacteria species used for this test include

clinical sample of Escherichia coli, Staphylococcus aureus and Klebsiella pneumonia. The

antibacterial activities of the compounds were estimated on the basis of the size of the

inhibition zone formed around the wells on sensitivity media. Antifungal activity of each

compound was determined using culture of three fungi species; they are Aspergillus niger,

Aspergillus flavus and Rhizopus species. They were cultured on potato dextrose agar. The

plates were incubated aerobically at 28±2°C for 96 h.

Treatment of Animals

Male albino rats (Wistar strain), weighing between 160 - 180 g were obtained from the

Department of Biochemistry, University of Ilorin, Ilorin and housed in the animal house of

the Department of Chemical Sciences, Ajayi Crowther University, Oyo, Nigeria for

acclimatization. They were kept in wire meshed cages and fed with commercial rat chow

(Bendel Feeds Nigeria Ltd) and water ad libitum.

Eighteen rats were divided into three groups of 6 rats per group. The first group was

used as control and received distilled water. The second group of rats was treated with free

ligand (mefloquine), while the third group was treated with metal complex (Co(Mef)Cl2).

The distilled water, ligand and solution of metal complex were administered orally to the

rats of various groups two times daily, morning and evening for seven days at the dose of

0.60 mg/Kg body weight. The animals were sacrificed 24 h after the last treatment.

Preparation of Serum and Tissue Homogenate

The method described by Yakubu et al.10

was used to prepare the serum. The rats were

sacrificed by stunning. Blood samples were collected by cardiac punctures into clean, dry

centrifuge tubes after which they were left for 10 min at room temperature. The tubes were


then centrifuged for 10 min at 3000 x g in an MSC (Essex, UK) bench centrifuge. The clear

supernatant (serum) was aspirated using a Pasteur pipette into clean, dry sample bottles and

then frozen overnight before use. The liver and kidney excised from rat, blotted of blood

stains were rinsed in 1.15% KCl and homogenized in 4 volumes of ice-cold 0.01 mol dm-3

potassium phosphate buffer (pH 7.4). The homogenates were centrifuged at 12,500 x g for

15 min at 4 °C and the supernatants, termed the post-mitochondrial fractions (PMF) were

aliquoted and used for enzyme assays.

Determination of Serum and Tissue AST, ALT, and ALP Activities

Serum and tissues AST, ALT and ALP activities were determined using Randox diagnostic

kits. Determination of AST and ALT activities were based on the principle described by

Reitman and Frankel (1957). ALP activity determination was based on the method of

Wright et al. (1972). The yellow coloured p-nitro phenol formed was monitored at 405 nm.

Protein determination of serum and all fractions was estimated by the method of Lowry et

al.13

as modified by Yakubu et al. and Malomo et al 10,21

using bovine serum albumin as

standard.

Statistical Analysis

The data were analyzed using one way ANOVA followed by Duncan multivariable post-

hoc test for comparison between control and treated rats in all groups. P values less than

0.05 were considered statistically significant.

Antimalarial Activities

The method used by Obaleye et al,14

was employed with slight modifications.

Nine Swiss mice were divided into three groups with three rats per group. They were

inoculated with 0.2ml of 1x106

parasitized erythrocytes suspended in buffered

physiological saline (pH 7.4). After 96hours of infection, the degree of parasitaemia was

determined from Glemsa stained thin blood smears by examining 100 erythrocytes in 5

different fields. This was expressed as a percentage of cell parasitized 14

. On the same day,

the drug was administered to the mice. A solution of ligand and metal complex was

prepared with a concentration of 1ppm each. 0.4ml of the solutions was injected into the

mice in each group, while only physiological saline solution was given to the control

animals. The mice were left for another 4 days after which a blood smear was prepared to

check the level of parasitaemia.

Results and Discussion

The metal chloride salt reacts with the ligand, L (L = Mefloquine) forming a compound

([M(II)LCl2]) using the proposed equation:

MX2 + L → ML .X2

Where M = Co2+

metal salt L = mefloquine and X- = Chloride ion.

The complex synthesized was found to be a non-hygroscopic solid with a peach colour

(Table1). The complex is very soluble in ethanol, methanol and distilled water. It has a


2249

sharp melting point, and no decomposition observed. The average percentage yield was

62.0%.The retention factor (Rf) values were calculated from the developed single spot for

the complex indicating the purity of the compound9. The Rf of the metal complex was

found to be higher than the ligand. Comparing the conductivity of the ligand with that of

the metal complex at a room temperature suggests that it is non-electrolytic in nature. The

analytical data of the anti-malarial metal complex showed 1:1 stoichiometry. The UV-spectra of the ligand and its metal complex have been interpreted in terms of

charge transfer transitions from the metal to the anti-bonding orbital of the ligand and of the

π → π* transitions of the ligand15

. The ultraviolet spectrum of the free mefloquine HCl

shows two absorption bands at 272.0 nm and 207.0 nm (Table 2). These transitions involve

energies of 36765 cm-1

and 48309 cm-1

. The bands have been assigned to the n→ π* and n

→ σ* transition respectively. These bands undergo hypsochromic shifts in the metal

complex due to Complexation. The infrared data (Table 3) showed the results of the most

informative and indicative region. The assignments have been interpreted based on

literature values obtained for similar structural compounds14

.

The shifts observed in the absorption bands between mefloquine and its metal

complex showed that there is coordination. Metal-Ligand bands were observed in the

ranges of 610 - 950 cm-1

in the metal complex. The Co (II) complex shows a µeff value of

3.5 BM, which corresponds to high spin (octahedral) stereochemistry16

.

Table 1. Some physical properties of mefloquine and its metal complex.

Compounds Melting point (°C) Colour

%

yield

Conductivity

(Ω-1

cm-1

dm-3

)

Mefloquine(Mef)Co(Mef)Cl2 259 - 260

242

White

Peach

-

62.0

-

1.287 x 10-4

Table 2. Ultraviolet/Visible spectral assignment of mefloquine and its metal complex.

Compound Wavelength

(nm)

Wave number

(cm-1

)

Mefloquine(Mef) 272.00

207.00

36765

48309

Co(Mef)Cl2

317.0

284.0

222.0

207.0

31546

35211

45045

48309

Table 3. IR spectral assignment of mefloquine and its metal complex.


Mefloquine

(cm-1

)

Co(Mef)Cl2

(cm-1

)

Tentative assignment

3447.4 w, b 3376.8 b V(OH), ν(N–H) stretch

2925.1 s, b 2942.5 w, b V(C–H) stretch of CH3

1586.2 s 1590.0 s V(C=N)

1380.9 s 1355.2 s V(C–N) stretch

Table 4. Magnetic moment of the ligands and metal complexes.

Compound Empirical formula Formular

weight

eff

(BM)

% Metal content

found

(calculated)

Mefloquine C17H16F6N2O 414.80 - -

Co(Mef)Cl 2 Co(C17H16F6N2O)Cl2 544.80 3.50 8.90 (9.04)

Figs 1 and 2 show the results of antibacterial and antifungal activities of free

mefloquine and the metal complex. The studies of the ligand and its metal complex gave

the antimicrobial activity of the compounds. The Metal complex was found to be more

active at higher (1.0 g/dm3) concentration than its corresponding ligand. The synthesized

complex was active against the three bacteria used, while they were found to be active

against only two of the fungi used, Aspergillus niger and Aspergillus flavus. Reports have

shown that CoCl2.6H2O has no inhibitory activity on bacteria and fungi species14

.

Figs 3 - 5 show the results of ALT, AST and ALP activities of the serum, kidney and

liver of rat. There was a significant increase (p<0.05) in serum ALT, AST and ALP

activities of mefloquine and its metal complex treated rats compared with the control, with

the mefloquine group higher than the metal complex. The data also indicate that there was a

significant reduction (p<0.05) in the liver and kidney ALT, AST and ALP activities of

mefloquine and its metal complex treated rats compared with the control, with the

mefloquine group lower than the metal complex. The observed significant increase in the

serum ALT, AST and ALP activities with a concomitant significant reduction in the same

enzymes activities in the liver and kidney of rats administered with mefloquine and the

metal complex may be as a result of stress imposed on the tissue by the drug, which may

lead to loss of the enzyme molecule through leakage into extra-cellular fluid. ALP is a

membrane-bound enzyme often used to assess the integrity of the plasma membrane and

endoplasmic reticulum20

. AST and ALT are enzymes associated with liver parenchymal

cells. They are raised in acute liver damage. They are also present in red blood cells, heart

cells, muscle tissue, pancreas and kidneys. When body tissue or an organ such as the heart

or liver is diseased or damaged, additional AST and ALT are released into the bloodstream.


2251

Both ALT and AST levels are reliable indicators of liver damage. In short, increase in

serum ALT and AST has been reported in conditions involving necrosis of hepatocytes18

,

myocardial cells, erythrocyte and skeletal muscle cells19

. Alteration in serum/tissue levels

of AST, ALT and ALP as recorded in these studies are indications of derangement in

cellular activities and hence no toxicity in both metal complex group and the mefloquine

group.

Fig 6 shows the antimalaria activities of the metal complex. Co (II) complex has the

best clearance with 80% reduction in parasitaemia.

0

5

10

15

20

25

30

35

40

45

E.Coli S.Aureous K.Pneumonia

% Z

on

e o

f In

hib

itio

n(m

m)

MEF

Co-MEF

Figure 1. Inhibitory activity of the ligands and metal complexes against Escherichia coli;

Staphylococcus aureus, and Klebsiella pneumonia.

0

10

20

30

40

50

60

70

80

90

100

A.Niger Rhizopus SP A.Flavus

% Z

on

e o

f In

hib

itio

n(m

m)

MEF

Co-MEF

Figure 2. Inhibitory activity of the ligands and metal complexes against Aspergillus niger,

Rhizopus species, and Aspergillus flavus.


0

20

40

60

80

100

120

140

160

180

Kidney Liver Serum

AL

P A

cti

vit

ies n

M/m

in/m

g P

rote

in

Control

MEF

Co-MEF

*

Figure 3. Effect of administration of ligands and metal complexes on the activities of

alanine amino transferase (ALP) of rat serum, kidney and liver.

* Significantly different from the control (p<0.05).

0

10

20

30

40

50

60

70

80

Kidney Liver Serum

AL

T A

cti

vit

ies n

M/m

g/P

rote

in

Control

MEF

Co-MEF

*

*


aspartate amino transferase (ALT) of rat serum, kidney and liver.


0

10

20

30

40

50

60

70

80

Kidney Liver Serum

AS

T A

cti

vit

ies n

M/m

in/m

g P

rote

in

Control

MEF

Co-MEF

**

*

*

*

*


alkaline phosphatase (AST) of rat serum, kidney and liver.



2253

0

50

100

150

200

250

Ave

rage

% paras

itaem

ia in

mice be

fore trea

tmen

t

Ave

rage

% paras

itaem

ia in

mice after trea

tmen

t

% R

educ

tion in paras

itaem

ia

% P

arasit

aem

ia

Chloroquine

Mefloquine

Co-MEF

Figure 6. Percentage Parasitaemia Of Ligand and Metal Complex Using Mice infected

with Plasmodium yoelii nigeriensis.

Conclusion

The results of the chemical and physical analysis from this study show that the ligand

(mefloquine) employed in this work coordinated with Co (II). The metal complex possesses

better physical properties than the parent compound. The toxicological studies revealed

metal complex showed mild toxicity particularly on the liver and kidney. Antimalaria

activities also revealed 80% clearance in parasitaemia reduction. Therefore it is evident that

the metal complex would be a better therapeutic drug for treatment of malaria. This

approach might be useful in Pharmacological research in solving the problem of drug

resistance in malaria chemotherapy.

Aknowledgement

The authors appreciate the financial support of Science and Technology Education Post

Basic (STEP B), University of Ilorin and Ajayi Crowther University, Oyo.

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